Mithramycins and process therefor

ABSTRACT

Mithramycins A, B, and C, methods for their production by fermentation using Streptomyces argillaceus, a new species of Actinomycete, or a new strain of Streptomyces plicatus, and methods for their recovery and separation are described. The products are of use as antimicrobial agents.

United States Patent [1 1 Sobin et al.

[4 1 Sept. 16, 1975 MITHRAMYCINS AND PROCESS THEREFOR [75] Inventors:Ben A. Sobin, Manhassett, N.Y.;

John B. Routien, Lyme, Conn.; Koppaka V. Rao, Cambridge, Mass.; WilliamS. Marsh, Wanaque, N.J.; Aline L. Garretson, East Lansing, Mich.

[73] Assignee: Pfizer Inc., New York, NY.

[22] Filed: Aug. 13, 1968 [211 App]. No.: 752,197

Related US. Application Data [63] Continuation-impart of Ser. No.617,442, Dec. 19, 1966, abandoned, which is a continuation-in-part ofScr. No. 317,399, Oct. 15, 1963, abandoned, which is acontinuation-inpart of Ser. Nos. 39,572, June 29, 1960, abandoned, andSer. No. 696,676, Nov. 15, 1957, abandoned.

[52] US. Cl. 424/119; 424/123; 424/124; 195/80 [51] Int. Cl A6lk 21/00[58] Field of Search 424/119, 123, 124; 195/80 [56] I References CitedOTHER PUBLICATIONS Primary Examiner-Jerome D. Goldberg Attorney, Agent,or FirmConnolly and Hutz 5 7] ABSTRACT Mithramycins A, B, and C, methodsfor their production by fermentation using Streplomyces argillaceus, anew species of Actinomycete, or a new strain of Streptomyces plicatus,and methods for their recovery and separation are described. Theproducts are of use as antimicrobial agents.

15 Claims, 4 Drawing Figures PATENTED SEP 1 6 I975 sum 2 or 4 PERCENTTRANSMITTANCE Omw mZOmUIz Z mxkwzwJ m 3 0 0 h w m O00 O00 000- 00: Do00m; 000 Dow- PERCENT TRAN SMITTANCE PATENTED SEP 1 6 I975 sumuqmPercent Transmiliance g 3 08m Qua This application is acontinuation-in-part of copending application Ser. No. 617,442, filedDec. 19, 1966, which is, in turn, a continuation-in-part of applicationSer. No. 317,399, filed Oct. 15, 1963, which is, in turn, acontinuation-in-part of application Ser. No. 39,572, filed June 29,1960, and application Ser. No. 696,676,

filed Nov. 15, 1957, all of which are now abandoned.

Background of the Invention This invention relates to the new and usefulfermentation products called Mithramycin A, Mithramycin B,

and Mithramycin C, to the salts thereof, and to mix- 1 5 tures of saidproducts and their salts, to their production by fermentation, tomethods for their recovery and concentration from crude solutions, suchas fermentation broths, and to processes for their purification. The

invention includes within its scope these products in dilute forms, ascrude concentrates, and also the pure crystalline forms thereof. All ofthese novel products are useful in combatting microorganisms, especiallyvarious Gram-positive microorganisms. in addition,

they are useful asdisinfectants against such microorganisms and they areuseful as an aid in the purification of mixed cultures for medicaldiagnostic and biological research purposes.

Srreptomyces [)liCtlIllS Parke-Davis & Company, Culture Bureau, Detroit,Michigan, No. 04918) is known 0 to produce three weakly basicantibiotics, amicetin, plicacetin and bamicetin (Haskell et al, J. Am.Chem. Soc. 80, 743-747 (1958), and British Patent No. 707,332). Theproducts of, the present invention, on

the other hand, are acidic substances. the properties of which aredescribed below. Y

SUMMARY OF Tl lE lNVENTlON (referred to as the mithramycin complex inour copending application Ser. No. 39,572) produced by the new strain ofS. plicatus is made up of about -98 percent mithramycin A and 15-20percent mithramycins B and/or C. These microorganisms were isolated fromsoil samples and identified by planting and testing cultures thereof onmedia normally used for such identification, while comparing theircultural characteristics with those described in Waksman andLechevalier, Actinomycetes and Their Antibiotics", 1953. Aculture of thenew species, Streptomyces argillaceus, has been deposited with theAmerican Type Culture Collection, Washington, DC, under the number, ATCC12956, while a culture of the new strain of Streptamyces plicatus hasbeen deposited with the same culture collection under the number ATCC12957. It is identified in the culture collection of Chas. Pfizer & Co.,Inc. as Isolate BA-l 1028.

The cultural characteristics of the new species, Streptomycesargillaceus, are set forth in the. following table, wherein thedescriptiongiven is based upon readings made after two weeks of growth.Classification of this new species was made by ,Dr. John 'B.Routien, whoproposed thename Streptomyces argillac'eus. The culture ATCC 12956 hasbeen designated the type culture of this new species.

Table I Streptomyces argillaceus sp. nov.

ATCC No. 12956 Amount of Aerial Mycelium Soluble Medium Growth andSporulation Pigment Remarks Pridham's Chains of spores straight Yeastorwavy or spirals at tip, Dextrose many chains in clusters; Agar sporesalmost spherical to oval to cylindrical averaging 1.6-2.01 X 1.0-1.3 p,but varying from 1.3 X 1.3 p.10 2.0 X 1.0 Glucose Poor or Almost none;None Vegetative mycelium Asparagine poor to gray-white to yellowish tanto brown; Agar moderate dark olive marginal area merulioid;

. reverse yellowish tan to brown. Skimmed Poor or Sparse; DrabVegetative mycelium Milk r to yellowish yellowish tan to dark brown;

moderate white milk coagulated, no

ptonization in one tube,

two other tubes had no coagulation or hydrolysis; pH change from 6.4 to5.7. Glucose Moderate Sparse to Dark Vegetative mycelium Agar moderateor blackish yellowish-grayish tan; moderate on brown reverse yellowishtan lower portion of to brownish black. slant; pale gray Nutrient PoorRarergray- Yellowish Vegetative mycelium Agar white brown colorless;reverse i 1 colorless. Synthetic Moderate Sparse to None Vegetativemycelium Agar moderate; colorless; reverse grayish-white colorless.Calcium Moderate Moderate; Sli ht Vegetative mycelium Malate pale gray Yyel owish colorless where visible;

1 becoming darker soluble reverse grayish tan; malate pigment digested.

. Table 1-.- Continued streptornyces ar gillaceus ATCC'NO; [2956 Amountof Aerial Mycelium Y Soluble V g Medium Growth and Sporulation Pigment vRemarks i 1 J V; .JIN Cellulose Moderate Good; No n e; '1 I brownishgray- Potato 4 Moderate Very sparse to Dark olive Vegctativemycclium Z IV Slants. i .to good 1; moderate (on one. brown dark olive-black; v 4 ntubc) reverse darkr a 1 olive-brown. 5 1 Starch Sparse ;.Good; grayishNone vVegetative mycelium Plates white to v colorless where visible; 5 Hv:

l dark gray reverse creamy whiteto 4 I T/ yellowish gray; partiall} 1*F. t v 11 f I 1 Y a it hydrolysis (zone of 1. a

I -hydrolysis 2.5 cmj'in ci L diameter). H Gelatin Moderate Good;.grayto A Dark- Vegetative myceliuni not 1 V Plates l grayish-white visible;colonies l i I f I depressed, marginalarea .Z smooth,lobed.-mor eor*less concentrically zoned; reverse dark'blackish q a brown; noliquefaction. =2 l Dextrose- Sparse Sparse; White Yery pale i,Vegetative mycelium L y v Nitrate" l yellow creamy white; no reductionBroth n ofnitrates to hitritesp "3 a. Emerson's; Moderate Sparse to DarkI Vegetative myceliu I I t I Agar moderate; Brown colorless where viblef whiteto E pale gray.

The cultural characteristics of the mithramy ci'nproducingfcultureIsolate'BA-l 1028 previously iden't i-' fied as a "hcw strain'ofStrepmmyces tanaslziensis, are described in Table 11 which presents a'side by-side comparison with S. plicatus'(Parke DaViS strain 049 lagartlm. S'ys'. Ba Jr. Sys. Ba e atinxoogabp 8t mi; Jr. 1 4 73: 15 27-,'1957'), kim'inedwing' (was ggpau'c't ist'ajr ch agar (1'20 gm! starch,7.0, distille 'd'HgO to" "lit )"waks'mansstarch agar: A waksmang rheActinornycetqlr, i 95o ,'f'b s 1 medium plus" va'riousS sources mister &Williams, Applied Mi; crobiology l2,' 46 521964); dextrose nitrate'bro'th (Waksman, The y tctinbmycets," 19:50), org nic nitrate broth(Gordon & Mihm, J. Barr cz 'kskwaksman, The Actinomycetes', 1950),glucose-asparagine agar (Waksm'an, The Actinomycetes, I950), nutrientagar Waksrnan, The Actinomycetes, 1950), and potato plugs (Waksman, TheActinomycetes, l950). Methods A were those given in the Int. Jr. Sys.Bact. paper of 1966.

Temperature'of incubation was 28 C. The results are assembled in TableII.

Medium S. lanasltiensis Srrepmmyces plicatus, ATCC N0. l2957 S.plic'atus BA- l l 028 Tyrosine Agar I Tryptone yeast extract brothDextrose nitrate broth Organic nitrate broth Skimmed milk MelaninMelanin l\litrites produced Nitrites produced Coagulation,

No melanin; No melanin pink soluble pigment after 4 days growth I onmedium.

No melanin No melanin Nitrites produced Nitrites produced Nitritesproduced Nitrites produced Coagulation slow Coagulation slow and r;),carbohydrate utilization 81,

Gelatin ",8 production (Lead acetate strips used for detection) Starchagar Waksmans Starch agar , pep'tonization; tan soluble pigment Iliquefaction {Strong from cysteine, Na ii O,

, and try'ptophahe and from peptoneiron agar plus yeast extract Nohydrolysis Strong hydrolysis andpartial. some peptonization, slight pinkto tan soluble pigment :Good liquefaction Strong in cysteine.

proteose peptone, Na- S O peptone and peptone-iron agar-plus yeastextract Very narrow zone Very narrow zone partial, some peptonization.pink to tan soluble pigment Good liquefaction Same as S. plic'alusexcept no H S from Na s O Narrow zone Strong hydrolysis Table IIC0ntinued Slreptomyces plicalus. ATCC No. 12957 Medium S. lunashiensisS. plicalus BA-l i028 Carbon Utilization Glucose yes yes yes Arabinose2+ 2+ Dulcitol Fructose 2+ lnositol 2+ lnulin Lactose 2+ 2+ 2+ Maltose2+ 2+ Mannitol 2+ Rafiinose Rhamnose 2+ Na acetate Sorbitol i Starch 2+2+ 2+ Sucrose Trehalose 2+ 2+ 10 Xylose 2+ 2+ Waksmans Starch agarAerial gray gray gray mycelium Reverse dark gray light brownyellowish-brown Soluble light brown lacking lacking pigment Inorganicsaltsstarch Aerial gray gray gray mycelium Reverse gray tangrayish-olive Soluble lacking lacking lacking pigment Yeast extractmaltextract Aerial gray gray gray mycelium Reverse light brown light browndark gray Soluble dark brown lacking lacking pigment Synthetic Aeriallacking scant; gray scant; gray mycelium Reverse colorless white whiteSoluble lacking lacking lacking pigment Nutrient agar aerial gray whitewhite mycelium Reverse pale tan cream cream to brown Soluble light brownlacking lacking pigment Glucose asparagine agar Aerial gray scant; graylacking mycelium Reverse brown white yellow Soluble light brown lackinglacking pigment Oatmeal agar Aerial gray gray gray mycelium Reversebrown dark gray yellow Soluble gray-brown lacking lacking pigment Potatoslices Aerial lacking gray gray mycelium Reverse dark brown to yellowishto yellowish to black yellowish-gray yellowish-gray Soluble black verypale gray very pale gray pigment Microscopic features Inorganic chainsof spores chains mostly chains 50-75% salts-starch mostly straight RAtype, l07c RA, 25-50% Spira;

some curved; Spira; 10-50 lO-SO spores per Yeast extractmalt extractOatmeal agar mostly 50 chains mostly flexuous, some RA type sporessmooth by electron microscopy spores per chain chains mostly RA someSpira spores smooth by electron microscopy chain chains mostly RA typespores spiny by electron microscopy utilization; differences in reverseand soluble pigment on potato and several other media; difference inshape of chains of spores.

hydrate utilization; slight differences in color of reverse and ofsoluble pigment on some media; spiny spores. The slight difference inproduction of H 8 and of degree of starch hydrolysis seem minor, and theonly absolute difference in carbohydrate utilization is on rafflnosesThe greatest difference is in the surface markings of the spores, andseveral papers in the literature indicate that the type of marking maynot be absolute for some species.

Because of these facts and the fact that S. plic'atus and BA-l I028 areso similar, it is concluded that BA- 1 1028 is a strain of S. pliz'atus.

It is to be understood that the present invention is not limited to useof the aforesaid organisms or to organisms fully answering the abovedescriptions, which are given only for illustrative purposes. It isespecially desired and intended to include the use of naturallyoccurring or artifically induced mutants and/or variants, such as thosewhich can be produced from the described organisms by various means,including xradiation, ultra-violet radiation, treatment with nitrogenmustards, and the like. For example, a subculture of BA-l l028A whichhas been found to produce mithramycin is described below. It isidentified in the culture collection of Chas. Pfizer & Co., Inc. asIsolate BA-l 1028A.

BA-l 1028A is a culture derived from BA-l 1028 by plating out singlespores of the latter and selecting different types of colonies. One ofthose selected was designated BA-l 1028A. Since it produced mithramycin,the same compound produced by BA-l 1028, it was concluded that it wasderived from that strain and was not a contaminant.

It was also grown on the media described in Table II at the same time aswere the other three cultures. Though it did not look exactly like BA-l1028, it is concluded that it still should be designated as a variant ofS. pliculus.

The differences between BA-] 1028 and BA-l 1028A are given as follows:very weak reduction of nitrate to nitrite by the latter; H 5 producedfrom Na S O (as by S. plz'calus) by the latter; no growth on rhamnose.On raffinose and sucrose the growth was ifor BA-l 1028A compared withfor BA-] 1028, while on mannitol and sorbitol it was for BA-l 1028A andi for BA-] 1028. Additionally, BA-l 1028 showed more brown color in thereverse on several media than did BA-l 1028A.

We wish also to include any organism, regardless of its appearance orphysiological behavior, that may be developed by means oftransformation, transduction, genetic recombination or some othergenetical procedure, using a nucleic acid or an equivalent material fromthe herein described species, whereby it has acquired the ability toproduce the elaboration product here described or to carry on thebiochemical change infrared absorption spectra of the various productsof this invention.

FIGS. 1, 2, 3 and 4 show the spectra of mithramycin A, mithramycin B,mithramycin A (anhydrous) and mithramycin C, respectively.

DETAILED DESCRIPTION OF THE INVENTION This invention includes within itsscope processes for growing the microorganisms S. argillac'eus ATCC12956 and S. plicutus ATCC No. 12957. The cultivation of thesemicroorganisms preferably takes place in aqueous nutrient media at atemperature of from about 2434 C., and under submerged, aerobicconditions with agitation. Nutrient media which are useful for suchpurposes include a carbohydrate, such as sugars, starch, glycerol, cornmeal; a source of organic nitrogen, such as casein, soy bean meal,peanut meal, wheat gluten, cotton seed meal, lactalbumen, enzymaticdigest of casein. A source of growth substances, such as distillerssolubles, yeast extract, molasses extract residues, as well as mineralsalts, such as sodium chloride, potassium chloride, potassium phosphate,magnesium sulfate, and trace minerals such as copper, zinc and iron, mayalso be utilized with advantageous results. A particularly useful andpreferred medium is one containing dextrose, soy bean meal and potassiumphosphate. If excessive foaming is encountered during fer mentation,anti-foam agents, such as vegetable oils, may be added to thefermentation medium. The pH of the fermentation tends to remain ratherconstant, but if variations are encountered, a buffering agent such ascalcium carbonate may also be added to the medium.

Inoculum for the preparation of the mithramycin products may be obtainedby employing growth from slants of the aforesaid microorganisms on suchmedia as Emersons agar or beef lactose. The growth may be used toinoculate either shake flasks or inoculum tanks, or alternatively, theinoculum tanks may be seeded from the shake flasks. The growth of themicroorganism usually reaches its maximum in about 2 or 3 days. However,variations in the equipment used, aeration, rate of stirring, and soforth, may effect the speed with which the maximum activity is reached.In general, the fermentation is continued until substantialantimicrobial activity is imparted to the medium, a peroid of from about24 hours to about 4 days being sufficient for most purposes. Aeration ofthe medium in tanks for submerged growth is preferably maintained at therate of about /2 to 2 volumes of free air per volume of broth perminute. Agitation may be maintained by means of agitators generallyfamiliar to those in the fermentation industry. Aseptic conditions must,of course, be maintained throughout the transfer of the microorganismsand throughout their growth.

After growth of the microorganism, the myeelium may be removed from thefermentation broth by filtration, centrifugation, or the like.Thereafter, the mithramycin products. may be recovered by severaldifferent procedures. For example, the filtered broth may be used as isor it may be dried. Preferably, however, the

products are further purified. For example, they may be extracted fromaqueous solution atsubstantially neutral to acidic pH conditions, i.e.pH of about 2 to 7, by means of a variety of water-immiscible organicsolvents, including the lower alcohols, aromatic alcohols,

esters, ketones, halogenated hydrocarbons and mixtures thereof. Examplesof these are n-butanol, sec.- butanol, amyl alcohol, hexyl alcohol,benzyl alcohol, ethyl acetate,'methyl isobutyl ketone, chloroform,ethylene dichloride and trichloroethylene. Of course. the solubility inthese solvents varies somewhat, the lower alcohols and methyl isobutylketone being preferred for best results. The mithramycin products may beextracted from most solvent solutions back into water by concentratingthe solvent extract and adding a nonsolvent for the antibiotic, such aspetroleum ether, cyclohexane, and the like. By adjustment of the pH tothe range set forth above, the product may be re-extracted into one ofthe wate'r-imiscible solvents indicated above. Upon drying the Solventand concentrating the solution, the product separates in crude form.

A more purified form of the mithramycin products is obtained bychromatographing a concentrate resulting from solvent extraction withone of the solvents indicated above, such as methyl isobutyl ketone orbutanol. The extract is preferably chromatographed on Florisil (anactivated magnesium silicate available from the.

Floridin Co.) or on an acid-washed alumina column which is thendeveloped with suitable solvents toisolate the mithramycin products. Thefollowing flow sheet shows a suitable method of recovering themithramycin products from the fermentation broth produced by each of themicroorganisms previously described:

S. argilluc'euus S. pliculus Fermentation Fermentation Filter BrothFilter armh Adjust Filtrate to Ac'd pH Adjust Filtrate to pH (5-7)Solvent Extraction (methyl isobutyl ketone Concentrate ExtractConcentrate Extract Alumina Column Wash with isopropyl ether Developwith CHCL -MEOH Filter Evaporate to Dryncss Acidify Filtrate Add Waterand Dry M ithramycin Products Alumina Column Elute with Methanol- EthylAcetate Dry-Eluate Mithramycin Products When the mithramycin mixture isproduced with S. urgillaceus, the alumina column, with the productadsorbed thereon, may be developed with a chloroformmethanol system.Upon evaporation of the eluate, addition of water and drying, a mixtureof pure microcrystalline mithramycins is obtained.

able solvent, such as methanol-ethyl acetate, results in a mixture ofpure microcrystalline mithramycins A, B and C.

The substance referred to in our first filed application, Ser. No.696,767, as PA 144 is identical with the substance referred to in Ser.No. 39,572 as mithramycin complex and referred to in Ser. No. 317,399 asa mixture of mithramycins A and B. In this mixture the proportionattributable to B is now sometimes found to consist of mithramycin Band/or mithramycin C. Mithramycin B wasnot described in our first filedapplication. It is produced in relatively minor amounts compared tomithramycin A and mithramycin C and in some instances may not beproduced at all or only in Solvent Extraction (butanol) SolventExtraction (Ethyl acetate) very small amounts. lt possessessubstantially the same biological activity as mithramycin A, thepredominant component, and the mithramycin mixtures produced herein. ltappears to be chemically of related structure to mithramycin A in viewof the similarity in physical propertiesof the two substances.Mithramycin C was not described in our earlier filed applications. itpossesses less biological activity than does mithramycin A ormithramycin B although it appears to structurally relate to both thesematerials. lts behavior on paper chroi Fraction l Frac-jon ll matographyis very similar to that of mithramycin B, so similar in fact that thetwo cannot be distinguished by this method. However, despite this, bothmithramycin B and mithramycin C are distinct substances and aredifferent from the materials to which reference was made in our earliestapplication Ser. No. 696,676 as Fractions A and B and referred trainSer. No. 39,572 as Fractions l and ll. The latter substances have only afraction of the biological activity of the mithramycin mixture.

A recovery procedure particularly adapted to recovery of themithramycins from fermentation broths is represented schematically inthe following flow sheet. The method involves absorption of the activeconstitucnts on activated magnesium silicate on Fuller's earth andelution therefrom. When employing Fuller's earth as absorbent, acetoneor ethyl acetate may also serve for elution.

Extract at pH 410 withzEthyl'acetatc= Raffinate Discarded Ethyl acetateConcentrate i a I to Purification The' ethyl'ace'tate cnc'hnatecontaining the mixture of mithraihycins'can then be further'purified, ifdesired or neces s ary,'by colunin chromatography' or by mg tercurr'entdistribution employing the solvent system 3" percent phosphate buffer pH7.0-7.5: ethyl acetate in an apparatus providing for 200 transfers. Theactive material concentrates intu be's' 80-160 and is'lrecov'eredtherefromby "concentration (if the combined ethyl ace tate phase,treatment of the concentrate with ether and extraction with water, Theaqueous extract is then frozen and dried from the frozen state.

Pure crystalline mithramycin A, mithramycin B and mithramycin C can beseparated from the crude ethyl acetate concentrate from the magnesiumsilicate or Fullers earth recovery procedure by chromatography cm.

Table 111 "acetate or acetone.

employing i1 silicica cid or a silicic aci'd-ce 'll 'ulose powdercolumn.Elution of this comma 'first with 'c't'hyl a ce ta t e followed by ethyl acetate cQntainin' gS percent volume of methanol providesin thefirstfraction', virtu cin s"B and'C'are held more tehaciouslybythe adsorbent and are'elutedonly when the methanol ethyl ace- -tatefsolventri'1ixture is'emplo'yed, The pro'du cts'ar'e recove'red fromfth eappropriate clu'ate fractions by concntr'atibn and crystallization frombut yl acetate, amyl The mith ramycin' A thusobtai'ned is a dihydrate.It is converted" to the anhydrous form by dr'ying"'a't C.

{oi- 24- ho'u'rs over phosphorfi's pentoxid'e under reduced .pr e s'sur'e at"0.05 mm. of "mercury.

The molecular formula of rn'ith'rarnyci'n A calculated from thefollowing analytical 'data ont'he anhydrous 'H'ydrOIySisKOilNl-ICL)"ofmithramycin' A shows it to be composed of an agly'cone (C HQOg), threesugars of the physical properties- 5f mithr'amy'cin A, hydrated andanhydrous forms, mithra'mycin A sodium salt and mithramycins B and C.Table IV contains a listing of color reactions observed with mithramycinA. Significant differences'in the infrared absorption of mithramycinsAjmd B occur in the region 1228 to 1720 Physical Properties MithramycinA Mithramycin B Mithramycin C Property Mithramycin A Sodium Salt(Anhydrous Form) Appearance Yellow Bright yellow Yellow 7 Orange yellowBright yellow crystalline long crystalline 'inicrocrystallinecrystalline solid solid rectangular solid solid prisms 1 Melting184-187" 270-275 dec. l84l87 l7075 dec. I l82-84 Point(C.) Behavior Dullyellow Bright yellow Brilliant yellow under ultra fluorescencefluorescence fluorescence violet light i I Ultra-violet 230 mu E, 240 muE, 1 83 230 mu E, Y i 230 mu E, 190 230 mu E, 220 10 220 10 220 I0spectrum: 278 mu E, 285 mu E, 475 278 mu E, 290 mu E, 312 280 mu E 55025 480 20 500 20 (Methanol) Ultra-violet 240 mu E, 230 mu E, 177 230 muE, 210 spectrum: 285 mu E, f" 289 280 mu E, 286 280 mu E, 294 (Water)400 mu E, 102 400 mu E, 97 440 mu E, 62 Analysis (3) C, 55.1 (b) C,54.65 (c) C, 57.70 C, 55.22 C, 55.25

H, 7.2 H, 6.95 H, 7.2 H, 7.13 H, 6.99 O, 37.3 0, 35.1 V 0, 37.65 0,37.76 (by difl'eren'ce) (bydih'erencef "i= (by difference)(by,difi'erence).fif W Equivalent 1115115 1 f Weight I I v v v iSpecific "--58 38.8-' 1' 1 Rotation PL: l 1 11.5. Methanol c=] a v l I 1lnfrared 736, 763, 803, y 5 806,852, 908, 722, 743, 808, 848, 905, 980,

Spectra 847, 901, 950, j 952, 983, 1004, 847, 903, 948 1020, 1070, 1166,V (0.03% 'in" 978, 1000, 1071, l l26','ll70, 978', 1000, 1063,123311266, 1293 KBr) 1064,1118, 1236, 1266, I300,v 1116, 1160,1228,.l408;;l5l5, l63.'

a 1160, 1228, 1330 1374, 1,515,. 1260, 1322,1347, 1706. 3436'cm.

(a) Calcd. for c,,H,,.o,,.2H,o;c,ss.70;1-1;7.19;o b difference), 37.10 i

I396, 1440, 1 500, 1580, I626, 1720; 2850, 2910,- 2950, 3390 cmf (seeH6. 1

(c) Calcd. for C52H1||0242 C, 57.55, H, 7.08; O (by difl'crence), 35.37

- (See FIG. 3

- .2s50.: 0, 2940, 3380 cm'."

1 (See .FIG. 2

' (seer-10.4

Table IV Color Reactions of Mithramycin A Property Mithramycin Alcoholicferric chloride Green Brick rcd prccipitato Couples to form a red color2,4-Dinitrophcnylhydrazinc Diazonium rcagcnt The mithramycins are acidicsubstances are readily form salts with bases, c.g. alkali andalkalineearth metal hydroxides and carbonates. They also form complexes withpolyvalent metal ions and are, therefore, useful for inactivating metalions, eg in biological experimentation and in metal separations.

Mild hydrolysis of mithramycin A with 210 percent acetic acid at 100 C.for 203O minutes or with 0.1NHC1 at room temperature overnight producesa product which appears to be identical to mithramycin C.

Mithramycins A, B and C may be assayed individually or as mixtures byobserving their activity against various tumors, such as Sarcoma 180 inmice and HS. No. l or CA. 755 in rats, in tests more fully describedhereinafter. However, a more convenient assay involves the use of themicroorganism B. subtilis as the test organism. A serial dilution typeof assay is employed, with the last dilution at which a distinct Zone ofinhibition occurs, being the number of dilution units/ml. of sample. Bycomparing this with the result obtained in the same test with a purifiedstandard, the potency of the sample is readily computed. In the assay ofmixtures, the C component, because of its lower biological activityrelative to that of mithramycins A and B, contributes relatively littleto the observed activity. The principal contribution arises frommithramycin A, the predominant component. The remainder of the observedactivity is due to the B and/or C components which may be present. Thecontribution of B when B is present usually outweighs that frommithramycin C.

A more convenient and reliable assay method comprises thin layerchromatography on silica gel with the system methyl ethylketonezmethanol:isopropanol (8:1 :1 In such an assay of a mixture ofmithramycins A, B and/or C, the separated mithramycin B and/or C spot isremoved from the plate, eluted with methanol and the optical density ofthe eluate read at 280 m,u.. Assay of four mixtures of mithramycins A, Band/or C isolated from S. plicatus fermentations by the procedure ofExample VII gave the following values for mithramycins B and/or C: 2.0,2.8, 3.8 and 4.8 percent. These values are representative for theisolated naturally produced mixtures As stated, the mithramycin mixtureis particularly active against Gram-positive organisms. The followingtable illustrates the antimicrobial spectrum of the mixture against avariety of microorganisms. These tests were performed under standardizedconditions in which nutrient broth containing various concentrations ofthe test material was seeded with the particular organisms specified,and the minimum concentration (MIC) at which growth of each organismfailed to occur was observed and recorded.

Table V Antimicrobial Spectrum of a Mithramycin Mixture (Approximately8571 A, 157' B and/or C) Table VI Antifungal Spectrum of a MithramycinMixture (Approximately 8571 A, 157! B and/or C) Hisluplusmu mpxulummTriclmp/zyton rubrum C'zuulidu alhit'anx No. 8

Allermlriu .Yoluni Axpz'rgillux Niger Penicillium funiculuxmn Claduspori um (Hornmdendrum clmlosporiuidex i indicates partialinhibition Activity against various resistant strains of Mycobacteriumtuberculosis was determined by testing the mithramycin mixture in Dubosmedium and incubating for a period of 14 days. The results of thesetests are set forth in the following table.

Table VII Activity Against Myeohactcrium tuberculosis Comparativeantibacterial activities for pure crystalline mithramycin A andmithramycin B appear in Table VIII.

Table Vlll Antibacterial Activity MIC meg/ml.

Microorganism Mithramycin A Mithramycin C penicillin and streptomycinpenicillin. streptomycin, tetracycline and urythromycin The mithramycinsare relatively toxic substances. A lethal dose in mice was found to beof the order of 2000 meg/kg. of body weight. The substance was lesstoxic by the subcutaneous route. In sevenday chronic toxicity tests onhealthy mice to which a purified microcrystalline mithramycin mixture(about 95% A 5% B and- /or C) was administered (two 0.5 ml. injectionsdaily of aqueous solutions containing appropriate drug concentrations),the LD was found to be 1500 mcg./kg.

Although mithramycin mixtures and the A, B and C components thereof maybe administered parenterally, eitheras an aqueous solution or' dissolvedin physiological saline, in the treatment of various infections inanimals, including human beings (as regards mithramycin A or mixtures ofA, B and/or C containing at least 85% A), various types ofpharmaceutical preparations may advantageously be compounded therewith.These preparations may include both liquid diluents suitable forextemporaneous preparations of solutions prior to administration.Illustrative of such diluents are: propylene glycol, diethylcarbonate,glycerol, sorbitol, etc. While other routes of administration arepossible, the parenteral routes are generally preferred. The variousdosage forms may contain buffering agents, as well as local anestheticsand inorganic salts to afford desirable pharmacological properties.

The techniques of isolated perfusion and regional perfusion,particularly the latter, have shown promise when preparations ofmithramycin products are used as the chemotherapeutic agent.

For most purposes, the solid preparations of mithramycin products, i.e.of mithramycin A, mithramycin B and mixtures thereof, should contain thecompounds in an amount of at least 0.05 mcg./mg. of the composition.Liquid preparations containing the active ingredient may be administereddirectly, e.g. by' syringe or, more desirably, by infusion. For directadministration an aqueous solution containing up to 0.5 mg. per ml. ofsolution is convenient. For infusion the active ingredicnt is desirablydiluted, for example, with isotonic glucose to one liter andadministered gradually over a 68 hour period. This latter methodeliminates or at least minimizes gastrointestinal symptoms which mayoccur on direct administration. The liquid preparations, such as aqueoussolutions, are particularly advantageous when the compound is employedin an amount of from about 0.1 to 2.5 mg./ml. of solution or suspension.

Preparations comprising pure mithramycin A or pure mithramycinB andmixtures thereof in any proportions can be used. The naturally occurringmixtures such as are produced by the herein described methods in whichmithramycin A is the major active ingredient as well as artificallyproduced mixtures in which the proportions of mithramycins A and B mayrange from substantially pure A to substantially pure B are useful. Froma practical standpoint, however, it is convenient to use the naturallyoccurring mixtures in which the proportions of mithramycin A to B and/orC range from about %987( to about 15 72 29 B and/or C.

The following examples are provided to illustrate specific processes forpreparation of the mithramycin complex and separation thereof into thepure crystalline A, B and C components. They are provided forillustrative purposes and are not intended to limit the scope of theinvention.

EXAMPLE I A nutrient medium was prepared from the following materials inone liter of water:

grams Glycerol l0 Corn Starch l0 Hydrolyzed Cascine (NZ amine B) 20Distillers Molasses Soluhlcs (Curhay 8.6. 5

The pH of the mixture was adjusted to 7 with potassium hydroxide and 5grams of calcium carbonate was added followed by steam sterilization forabout 30 to 40 minutes. A slant culture of the species S. argillaceusATC C 12956 was transferred to ml. of this medium in a 300 ml.Erienmeyer flask and shaken 4-5 days until good growth was obtained.lnoculum for a larger fermentation was prepared by transferring thecontents of the aforesaid flask under aseptic conditions to one liter ofthe same medium in a 3 liter flask and shaking the same for 48 hours.

Fifty gallons of nutrient medium having the composition indicated abovewere prepared. sterilized, and inoculated with the inoculum thusprepared. The organism was then cultivated under submerged conditions ofaeration for a period of 3 days. The fermentation broth was filteredwith the aid of Super Cel (diatomaceous earth) without adjustment of thepH. The filtrate was then adjusted to pH 2.0 with concentrated sulfuricacid and 10 percent sodium chloride was added per volume of thefiltrate.

The filtrate was then twice extracted with 5 gallons of methyl isobutylketone. The solvent extracts were separated, combined and evaporated invacuo to 500 ml. The resulting 500 ml. concentrate was nextchromatographed on an acid-washed alumina column prepared withchloroform. The column was developed with chloroform, followed by a 95percent chloroformmethanol system. and the major proportion of themithramycin complex was removed with a 90 percent chloroform-methanolsystem as a reddish-brown fraction.

Approximately 400 ml. of this fraction was evaporated in vacuo to 50 ml.and the resulting concentrate was again chromatographed on an acidwashedalumina column prepared with chloroform. The column was developed asabove. i.c. with chloroform, 95 percent chloroform-methanol. and 90,percent chloroformmcthanol removing the mithramycin mixture whichoccurred as a bright yellow band. One-third of the resulting cluate wasevaporated in vacuo to dryness. taken into water, filtered. and, onevaporating to dryness, yielded 223 mg. of microcrystalline mithramycinecomplex. The remaining two-thirds of the eluate was evaporated todryness, taken into water, filtered and freezedried, yielding furtherproduct in the form of a bright yellow powder.

EXAMPLE ll A culture of S. pliumix ATCC No. 12957 was cultivated in amannersimilar to that indicated in Example I. first in a 4-liter glasspot and then in a ISO-gallon tank using a medium of the followingcomposition:

grams/liter Glucose "Cerelose") l Soy Bean Meal l5 Distillers Solubles2.5 Sodium Chloride 2 Dipotassium Phosphate S Calcium Carbonate Water 1To one liter The medium was adjusted to a pH of 7. sterilized. andinoculated with 5 percent by volume of a 48-hour culture grown asaforesaid. The fermentation was continued for a period of 45 hours.

Upon completion of the fermentation, the broth was filtered with about 5percent Super Cel (diatomaceous earth 5 g./ l 00 cc. broth) and the cakewas washed with one-twentieth volume of water. A sample of the filtratewas found to exhibit a high order of antimicrobial activity. Thefiltrate was adjusted to pH 6.0 and extracted once with one-half volumeof n-butanol. The extract was concentrated in vacuo, with the additionof water to lower the distillation temperature, until it reachedone-half of the original volume. Further concentration, without theaddition of water, was carried out until one-fiftieth of the originalvolume was reached. The resulting concentrate was permitted to stand at5 C. for hours. during which time it deposited a tan-coloredmicrocrystalline solid, herein designated as Fraction 1. This fractionwas separated by filtration and washed with butanol.

Physical Properties of Mithramyein Mixture and Fractions l and II Thebutanol filtrate resulting from the above extrac tion was thenconcentrated further in the presence of water to remove most of thesolvent. The dark colored residue which resulted was treated with 3percent acetic acid and extracted three times with equal volumes ofethyl acetate. It was found that all except about 5 to 10 percent of thetotal microbiological activity passed into the solvent layer. Theextract was concentrated to a small volume and added to a column ofacidwashed alumina in ethyl acetate. Approximately 15 to 20 grams ofalumina were employed per gram of crude solids in the extract, thesolids content being about 5 to l0 percent of the extract.

The initial fractions from the column showed no antibacterial activitybut on concentration yielded a colorless crystalline solid hereindesignated as Fraction ll. Thereafter, the mixture of mithramycins waseluted from the column with ethyl acetate, followed by 10% ethanol inethyl acetate. The active fractions were combined and freed from solventunder reduced temperature, whereby the mithramycin mixture was obtainedas a bright, amorphous powder.

EXAMPLE "I The fractions obtained by the procedure of Example II werefurther purified as follows.

Fraction I was crystallized from aqueous butanol, which, after twocrystallizations, yielded a colorless product in the form of hexagonalplates.

Fraction ll was crystallized twice from a mixture of pyridine andmethanol, separating out as colorless-needles.

Fraction llI (mithramycin mixture) was purified by warming with amylacetate. filtering and concentrating the filtrate. lt separated out as alemon yellow microcrystalline solid.

EXAMPLE 1V Another fermentation was conducted, as in Example ll, with amedium having the following composition;

grams/liter Glucose ("Cerclose") l0 Soy Bean Meal l5 ioiir'o 5 NaCl 2Distillers Solubles 2.5 CaCO;, '2 Water To one liter After adjusting topH 7.0 and sterilizing, the medium was inoculated with the variant of S.pliculus descibed in Table III. The filtered broth was found to beactive against Sarcoma I in mice, when tested as hereinafter described.

The mixture of mithramycins and the fractions that are producedtherewith are quite distinct from one another, as evidenced by a numberof their physical and chemical properties. Some of the more important ofthese properties are summarized in the following table.

Table IX Fraction II] Property Fraction l I 7 Fraction ll MithramycinMixture I Color white white lemon yellow Crystal Structure hexagonalneedles microscopic prisms plates Infrared Absorption cm'. cm cm" Maxima(in potassium bromide pellet). 9l0 720 BIO =Table lX-Continued F I"Physical Properties of Mithrumycin Mixture and Fractions-l and ll v I 4Fraction-Ill Property Fraction I II Fraction Mithramycin Mixture Colorwhite Whlt= lemon yellow; I I I Crystal Structure hexagonal l mroscopic'pris'ms' plates I I cm crncm" 975 I -755 905 1046 7x0 954. 1075790 982 H38 s20 I008" I175 843 I070 I258 864 .ll22 1295 90s "'1 170 1360922, 123 8 iv I385 965 .1265 1420 I I010 1300 -l615 1020 13351 2800.I050, 13 I I 3340 1100 I420 I I I m r 515 l l 15 I580 I l l 85 i630 I215I730 1490 I I 1530 I560 l580 I 1620 3400 Ultraviolet Absorption(Methanol Solution) Mnfl'" 1 rim MW'" rm 252 8.7 255 l 257 10.2 315 400263 9.1 385 I l.l 405 L0 Optical Activity [a],,"' [a],, (MethanolSolution, C=l.0) (Dimethylformamide, C=l .0) none none Melting Point 255300C. |-75C.' decomp. decomp. Analyses ,7,

Carbon 55.98 67.26 55.49 Hydrogen 8.17 4.53 I 7.613, Oxygen (bydifference) 35.85 28.2l X 3688 I I pK value V (in water) ';7 O ColorwithFerric Chloride none none dark gre en Reaction with v I diazoniumchloride negative bright red bright; red I Solubility I Slightly watei'lower most common 'diethylether, soluble in: alcohols, organicsolvents, benzene;chloroa E I, v

acetone water I form, amylacetate ethylene dichloride; trichlorethy Iene, I A soluble in: hot water and pyridine, water, lower alcohols,

mixtures of" dimethyl -i;e. 'methyl, ethyl t r waterand formamide, propyl,nbutyl,sec. 0 I lower alcohols, aqueousNaOH butyl, amyl, hcxyl I I I Ipyridine, illcohokacetonei *I' "-1-" r dimethyl ethyl acetate, methylformamide isobutyl ketone, aqueous NaOH benzyl alcohol insoluble in: Inonpolar organic I no n polar organic carbon tetrachloride.

solvents, i.c. solvents i.e. '8 petroleum ether, benzene carbonCarbontetrachlorcyclohexane tetrachloride, ide. benzene,

, petroleum ether petroleum ether potassium hydroxide, lithiumhydroxide, ammonium 1 hydroxide or the like, in aqueous solution orunder anmg to about pH 1.0 sodium'hydr'oxide and freeze drying hydrousconditions, For'exa'rnple, the sodium salt can be prepared by dissolvingthe base in water and titratthe resulting solution:- O ther relativelystrong bases may, of course, be employed to prepare the correspondingsalts.

EXAMPLE v An S. plicatus fermentation is conducted as described inExample II, the whole broth adjusted to pH 4.0, and the myceliumfiltered. An activated magnesium silicate (30-60 mesh) column (Florisil,Floridin Co.) is prepared containing 75 g. of absorbent per gallon offermentation broth. The filtered broth is then passed through the columnat a free rate of 3040 gal. per hour. After the entire batch of filteredbroth has been fed to the column, it is followed by water which washesthe broth in the column through. The water wash is dis continued whenthe effluent is colorless or when the total solids contained thereinfalls to about 1 mg./ml. The solids content of effluent broth is about20 mg./ml.

The column prepared in this fashion is then eluted with methanol.Fractions of convenient size are collected and their optical density at280 my. is measured. Those fractions having an optical density in excessof 2.0 are combined. A plate assay for activity against .S'laphylococcusaureus on each of these fractions is conducted.

The combined methanol eluate fractions containing active material arethen concentrated to dryness, dissolved in a solvent mixture made up ofthree parts by volume of ethyl acetate and one part by volume ofbenzene. A chromatographic column having a 2 V2 inch diameter and 15inch length is then prepared employing a 2:1 mixture of silicic acid andcellulose powder (weight basis) as packing using the same solventmixture in packing the column.

A portion of the ethyl acetate concentrate containing 5 to 8 g. of amixture of mithramycins is then added to the column and the columndeveloped with ethyl acetate collecting the eluate in 400 ml. fractions.Mithramycin A is rapidly eluted, traveling almost with the solventfront. The bulk of it is obtained in the first l O fractions (4 l.), butan additional fractions (6 1.), containing a small amount of additionalmaterial due to tailing, are collected. The eluting solvent is thenchanged to ethyl acetate containing 5 percent by volume of methanol.Mithramycin B is collected in fractions 60l00. The progress of columndevelopment is readily followed visually either in ordinary room lightor ultraviolet light.

The fractions containing mithramycin A and mithramycin B are separatelyevaporated; the residues crystallized from n-butyl acetate or amylacetate yielding the products in pure crystalline form. They exhibit theproperties tabulated in Table IV.

Alternatively, the pure crystalline sodium salts of mithramycin A andmithramycin B can be prepared by concentration of the combined eluatefractions to a small volume and extraction thereof with water,adjustment of the water extracts to pH 8.0 with sodium hydroxide,freezing of the alkaline extract, and drying thereof from the frozenstate. Other metal and organic base salts can be prepared in analogousfashion by sub stitution of the appropriate base. For instance, thecalcium, magnesium, zinc, aluminum, ammonium, ,B-hydroxyethylammonium,procaine, triethylammonium, ethylene diamine, potassium, lithium,ferric, and ferrous salts are thus prepared. The physical properties ofthe sodium salt of mithramycin A are listed in Table III.

EXAMPLE VI A mixture of mithramycins A and B can be convenientlyprepared in purified microcrystalline form from the methanolic eluateprepared as described in the first two paragraphs of Example V byconcentration thereof to a low volume resulting in removal from the bulkof the methanol from the solution and extraction of the concentrate withethyl acetate at pH 4.0. The ethyl ace tate extracts are then combinedand concentrated to a convenient volume for countercurrent distributionin a 100 tube automatic apparatus. They are distributed against 3percent phosphate pH 7.0 to 7.5 buffer. The contents of the tubes arecombined in groups of then and assayed by the B. subtilis plate assay. i

The bulk of the activity is found in tubes through 160. These fractionsare combined and concentrated to a small volume, ether is added theretoand the mixture is extracted with water. The water layer is freed ofsolvent by evaporation, frozen, and dried from the frozen state. Ayellow amorphous solid comprised of a mixture of approximately topercent mithramycin A and 5 to 15 percent of mithramycin B is obtained.

The pure crystalline sodium salt of mithramycin A is prepared from theaqueous extract described in the previous paragraph by adjusting it topH 8.0 with sodium hydroxide and drying from the frozen state. Theresidue is crystallized from methanol or ethanol. A crystalline productis collected and recrystallized from 1:] aqueous methanol yielding thepure crystalline dehydrated sodium salt as bright yellow elongatedrectangular prismatic crystals.

Occasionally, during the course of countercurrent distribution in thisfashion the sodium salts of a mixture of the mithramycins A and Bseparate in the tubes containing the highest concentration thereof. Thesodium salts are moderately soluble in water but only slightly solublein aqueous sodium chloride or sodium phosphate solutions. Theirsolubility in lower alkanols is very low and they are almost insolublein the common organic solvents. The physical properties of purecrystalline sodium mithramycin A are listed in Table III.

EXAMPLE VII An S. plz'catus fermentation is conducted as described inExample 11, the whole broth adjusted to pH 5 and the mycelium filtered.The filtrate is extracted with onethird volume n-butanol and the extractconcentrated azeotropically.- The concentrate is shaken between waterand isopropyl ether (1:1), the phases separated and the aqueous phasebrought to pH 7 with dilute so dium hydroxide solution. The neutralizedsolution is then freeze-dried and the residue extracted with ethanol.The ethanol extract is concentrated in vacuo to a thick solutionwhereupon the sodium salt of mithramycin A crystallizes and is filteredoff. The filtrate is concentrated, transferred to ethyl acetate from anacid solution (pH 23) and adsorbed on a silicic acid column. Elution ofthe column with 5% methanol-ethyl acetate 7 gives a mixture containingpredominantly mithramycin EXAMPLE VIII The organism, Strepmmycesplicutus ATCC 12,957, was grown in submerged culture in a mediumcomposed of the following ingredients in grams per liter of water:

Calcium carbonate A 2 to 3 day old vegetative inoculum was used to theextent of one to five per cent and the fermentation carried out forabout 4 days at 28 30 C. (The progress of the fermentation can befollowed by a Bacillus subtilis plate assay at a suitable dilution).

The culture liquid was filtered with the aid of l to 2 per cent HyfloSupercel at the existing pH (7.0 7.5). The filtrate was adjusted to pH5.0-5.5 and extracted once with n-butanol (25 per cent of the volume ofthe broth). The extract was separated, clarified and concentrated in thepresence of water until almost all the solvent was removed. Theconcentrate was then shaken with an equal volume of isopropyl ether toremove oily impurities, then extracted twice with ethyl acetate at pH2.0-3.0. The solvent extract was concentrated to a small volume andchromatographed on a column of acid-washed alumina (20 to 30 grams pergram of solids) made up in ethyl acetate. The sample was applied to thecolumn and the latter eluted first with ethyl acetate and then withethyl acetate containing increasing amounts of methanol. The bulk of theactivity was recovered in the to per cent methanol-ethyl acetate eluate.The compound was recovered by concentration of the active fractions andcrystallized twice from acetone. Mithramycin A separates as a brightyellow crystalline solid.

The homogeneity of the product was demonstrated in the I following thinlayer chromatography system: Methyl ethyl ketone-isopropanol-methanol(8:1:1) Silica Gel G. R 0.60.

Further solution of the column with percent methanol-ethylacetateaffords predominantly mithramycin C and minor amounts of mithramycin B.This product is purified by chromatography on a silicic acid column(made up in ethyl acetate) followed by elution first with ethyl acetateand then with ethyl acetate containing increasing amounts of methanol.The C component is recovered from the 5 to 10 percent methanolethylacetate eluate.

EXAMPLE IX The fermentation procedure of Example VIII is followed andthe products recovered by the following modified process.

The whole broth (approximately 1000 gallons) is filtered on .an Oliverrotary filter, the pre-coat of which is neutralized with caustic soda topH 6.0-7.0.

Fifty to one hundred pounds of Hyflo Supercel are added to the brothwith agitation before filtration on the Oliver.

The filtrate is received in a tank suitable for solvent extraction.After filtration is complete the pH of the broth is adjusted to 5.0-5.5with acid and saturated with n-butanol. The saturated broth filtrate ispassed over a Podbielniack separator against n-butanol at 3 to 1 ratio.

The spent liquor (raffinate) is collected in a suitable A vacuo at 30-35C. to remove the solvent, water being added throughout to aid in removalof the butanol. A solution of 0.1 percent dibasic phosphate buffer (Naor K) is added to the vacuum pan to neutralize the residual acids andconcentration continued until all the solvent is removed and the volumeis less than 10 percent of the original volume.

The concentrate is then washed with isopropyl ether to remove oilspresent, re-concentrated to remove the isopropyl ether then stirred with10-15 percent sodium chloride. The pH is adjusted to 2-3 with acid, andthe acidified concentrate extracted twice with ethyl acetate. The spentliquor (ethyl acetate raffinate) is then stirred with 5 lbs/50 gals.Hyflo Supercel and filtered.

The filter cake is suspended twice in methanol, filtration being carriedout after each suspension. The spent liquor (after filtration) isassayed, and then discarded if potency is low.

The ethyl acetate-methanol filtrates are concentrated in vacuo (30-35C.) then put on a Florisil column made up in ethyl acetate (15-30 gms.per gm. solid). The column is eluted with ethyl acetate, then with ethylacetate containing increasing amounts of methanol.

Most of the activity comes off in the 10-20 percent methanol fraction.The mithramycin A is then recovered by concentration of the activefractions, and crystallation of the solid obtained.

Activity in the broth, solvent extracts and column fractions can befollowed by biological assay with Bacillus subtilis or Slrepmmycesaureus. or by optical density at 280 mu.

The B and C components are isolated by the procedure of Example VIII.

EXAMPLE X Purification and Characterization of Mithramycin C Filteredbroth gallons) Extract with 25-35% n-butanol at pH 4-6 Rafi'matc Extract(discard) Concentrate in presence of water to 3-5 liters stir with NaClto give 207: concentration and Cake Extract with two 3- liter portionsof 25-50% methanol-ethyl acetate and filter Filtratc Cake (discard)Filtrate Extract once with 5 liters ethyl acetate v Raffinate Emma(discard) Combine and concentrate to about 2-4 liters The concentratefrom the above flow sheet is stirred with 500-800 g. of Florisil and theslurry is diluted with 2-4 liters of ethyl acetate. The slurry is addedto a column of Florisil (3-5 kg.) in ethyl acetate. The column is elutedsuccessively with ethyl acetate, 10, 20 and 40 percent methanol in ethylacetate. The first two solvent cluates contain inert substances as shownby lack of activity against B. subtilis. The 20 percent methanol-ethylacetate fraction contains almost essentially mithramycin A fraction. Thelast eluate contains the residual quantities of mithramycin A and thebulk of mithramycin C.

Concentration of the 20 percent methanol-ethyl acetate fraction to asmall volume 1.5-2.5 liters) yields a crystalline solid, the magnesiumchelate of mithramycin A. This can be recrystallized from methanol-ethylacetate i=3) for further purification.

The magnesium chelate crystallizes as large hexagonal prisms whichdecompose gradually at 2l0-2l5 C.

Analysis Calcd. for (C H O- UMgQH- O: C, 56.04; H, 6.96; Mg 1.09%;Found: C, 55.64; H, 7.19; Mg 1. l 5%.

When titrated in 50 percent aqueous methanol with hydrochloric acid, itshows a pK,, value of 4.5-4.7 and an equivalent weight of l l 10. Theequivalent weight calculated for (C H O Mg is 1114.

The crystalline magnesium chelate is converted to mithramycin A bystirring between water and ethyl acetate while adding hydrochloric acidto pH 3.0-4.0. The solvent layer is separated and the aqueous layerextracted twice more, preferably in the presence of 5-10 percent salt.The combined solvent layers are concentrated to dryness and the solid iscrystallized from ace tone. Mithramycin A separates as a bright yellowcrystalline solid from acetone.

Titration in 50 percent aqueous methanol with sodium hydroxide shows apK,, value of 7.6 i 0.2 and an equivalent weight of l l 15 i 15.(Calculated for C52H16O24-2H2O l The mother liquors from thecrystallization of the magnesium chelate are added to the 40 percentmethanol-ethyl acetate eluate of the Florisil column and concentrated toa small volume. This concentrate is shaken with acid water (pH 3-4) toconvert the sample to the free acid form. The solvent layer isconcentrated to remove most of the methanol and added to a column ofsilicic acid-cellulose (2:1) in ethyl acetate. About 15-25 g. of silicicacid is used per gram of the crude mithramycin as estimated by opticaldensity at 280 my. using the relationship 50,000 optical density unitsequal 1 g. The column is eluted with ethyl acetate, 3-5 percentmethanol-ethyl acetate, percent methanolethyl acetate. The progress ofthe column is followed by optical density and the solvents are changedas the need arises. Usually, mithramycin A appears with the ethylacetate and, if the band moves too slowly, one can use the 3-5 percentmethanol-ethyl acetate to speed its clution. The mithramycin C is elutedeither with the 3-5 percent methanol-ethyl acetate or 10 'percentmethanol. The fractions are tested by thin layer chromatography in thesystem: methanol-isopropanolmethyl ethyl ketone (1:1:8). Mithramycin Ahas an Rf of 0.6-0.7 while mithramycin C has an Rf of 0. l 5-0.25.Fractions which contain mostly the C component are combined,concentrated to dryness and crystallized from acetone. lts solubility issimilar to that of mithramithramycin A as shown below. The values shownbelow are the diameters of the zones in the disc assay method using B.suhtilis.

Concentration Mithramycin A Mithramycin C so 'y/cc 25.8 20.8 25 24.5l8.4 12.5 23.0 l6.8

Salts of mithramycin C, such as the sodium, lithium, potassium andammonium salts, are prepared according to the procedure of Example lV.

EXAMPLE XI Several fermentations were carried out with S. plicatus(Parke-Davis & Company, Culture Bureau, Detroit, Michigan, No. 04918)using the following medium.

gmslliter Glucose (Cerelose) l0 Com Starch l0 Hydrolyzed Casein (NZAmine B) 5 Distiller's Solubles 5 Sodium Chloride 5 Soy Bean Meal l5Calcium Carbonate l Water To one liter The mixture was adjusted to pH 7then sterilized. A plug from a slant culture of S. plicatus (Parke-Davis& Company culture No. 049l8) was placed in 200 ml. of this media in ashake flask and the flask shaken for three days. Two thousand ml. ofthis medium in a 4 l. pot was inoculated aseptically with 1 percent (20ml.) of the inoculum from the shake flask. A series of such pots was setup and fermentation run for periods of 24, 48, 72 and 96 hours.

Upon completion of the fermentation each of the broths was filtered andextracted with n-butanol at pH 5. The butanolic extractions wereconcentrated to near dryness. The concentrates were shaken withmethanol, the methanolic solution filtered then subjected to thin layerchromatography in the systems methyl ethyl ketonezisopropanol:methanol(8:1:1) silica gel G; and 0.02 N sodium chloride ondiethylaminoethylcellulose.

The methanol filtrate was chromatographed on Florosil as described inExample V and the column eluted successively with methanol-ethyl acetatein which the methanol concentrations were 5, 10, 20 and 40 percent,respectively. The eluates were combined, concentrated to near drynessand the residue taken up in methanol. The ultraviolet spectrum of theeluates showed maximum absorption at approximately 262 mg. Themithramycins, as noted above, exhibit maximum absorption in theultraviolet at 280 mu.

Thin layer chromatography of the concentrated eluates in the abovementioned systems, and paper chromatograms in the systems butanolzaceticacid2water (5:1:4) and butanol saturated with water showed nomithramycins were present. All chromatograms were diethyl ether andbenzene, and substantially insoluble in petroleum ether, carbontetrachloride and cyclohex- 'ane, which is capable of forming salts withbases, the

pure crystalline dihydrate of which is yellow and con tains the elementscarbon, hydrogen,'and oxygen in substantially the following percentagesby weight: Car- 5 bon 55.5%, Hydrogen 7.2%, Oxygen 37.3% (by difference)and has the molecular formula C H O .2H O; which displays ultravioletabsorption maxima in methanol solution at 230 and 278 m;:., which has anequivalent weight of substantially l 1 i 15 and a pK,, value of 7.6 i0.2, which exhibits a dull yellow fluorescence when irradiated withultraviolet light, and which as a 0.03 percent dispersion in potassiumbromide exhibits absorption maxima in the infrared region of thespectrum at 736, 763,803, 847, 901, 950, 978, 1000, 1064, 1118,1160,1228, 1259, 1293, 1320, 1370, 1396, 1440, 1500, 1580, 1626, 1720, 2850,2910, 2950, 3390, cm 1 and which when dissolved in methanol at aconcentration of 1 percent has the specific rotation 58; the crystallineanhydrous form of which is yellow, melts at l84l87 C. and contains theelements carbon, hydrogen and oxygen in substantially the followingpercentages by weight: Carbon 57.70%, Hydrogen 7.2%, Oxygen 35.1%, (bydifference) and has the molecular formula -C H ,,-O and which displaysultraviolet absorption maxima in methanol solution at 230 and 278 my.and which as an 0.03 percent dispersion in potassium bromide exhibitsabsorption maxima in the infrared region of the spectrum at 8,06, 852,90 8,

' 28 crystalline form ofiwhic h is orange-yellow and containstheclements carbon,hydrogen, and oxygen in substan tially the followingpercentages by 'weight: Carbon 55.22%, Hydrogen 7.13%, Oxygen 37.65%,(bydifference) which'displays ultra-violet absorption maxima in methanolsolution at 230, 290, 320 and 410 mu, which exhibits a bright yellowfluorescence when irradiated.

with ultra-vioilet light, and which as a 0.03 percent dispersion inpotassium bromide exhibits absorption maxima in the infrared region ofthe spectrum at 722, 743, 808, 847, 903, 948, 978', 1000, 1063, 1116,:1160, 1228, 1260, 1322, 1347, 1368, 1430, 1440, 1500, 1585, 1630, 11720,2850, 2900, 2940, 3380 cm. 4. ,A basic salt of the substancedefined inclaim 3. I 5. Mithramycin C,. an acidic substance solublein water,lower-alcohols,- ethyl acetate, acetoneand methyl1330,1374,1515,1631,1724, 2890 and 3413 cm..; and which on thin layerchromatography in the system methanol: isopropanol:methyl ethyl ketone1: 1:8) has an R, value of 0.6-0.7; the crystalline magnesium salt ofwhich is bright yellow, decomposes at 2l02l5.C., has a pK,, value of4.5-4.7 and an equivalent weight of l 1 l0, and contains the elementscarbon, hydrogen and magnesium is substantially the followingpercentages by weight: Carbon 55.64%, Hydrogen 7.19%, Magnesium 1.15%and has the molecular formula (C H O Mg; the pure crystalline dihydratedso dium salt of which is bright yellow and contains the 'elements carbonand hydrogen in substantially the following percentages by weight:Carbon 54.65%, Hydrogen 6.95% and has the molecular formula C5,2H O 4Na.2-' H O, which displays ultraviolet absorption maximain methanolsolution at 240, 285, 315, and 425 my. and when dissolved in methanol ata concentration of l per cent has the specific rotation +38.8. I w l I2. A basic salt of the substance defined in claim 1. 3. Mithramycin B,an acidic substancewhich is highly soluble in water, lower alcohols,acetone, ethyl acetate and methyl isobutyl ketone, slightly soluble indiethyl ether and benzene, and substantially insoluble in petro leumether, carbon tetrachloride, and cyclohexane, which is capable offorming salts with bases, the pure isobutyl ketone, slightly soluble indiethyl ether and benzene, and substantially insoluble in petroleumether, carbon tetrachloride and cyclohexane, which'is capable of formingsalts with bases, the pure crystalline 'form of which is bright yellow,melts at' 182l 84C.

and contains the elements carbon, hydrogen and oxygem in substantiallythe following percentages by 12957 in an aqueous nutrient medium underagitated submerged, aerobic conditions at a temperature of from about 24to 30C. for a period of from about one day. to about four days untilsubstantial antimicrobial activity is imparted .to such medium.

8. Theprocessclaimed in claiml'7 wherein a sub-.

stance selected from the groupconsisting of mithramycin A, mithramycin Band mithramycin C is recovered from the fermentation-broth by filteringthe broth, adjusting the filtrate to a pH of. about 6, extracting thefiltrate .withn-butanol, concentrating the extract, allowing theconcentrated extract to stand to effect crystallization of a portion ofsaid substance, removing the crystals byfiltration, concentrating andacidifying the filtrate, extracting the filtrate with. ethyl acetate,concen tratingthe extract, absorbing said substance from the extractonacid-washed alumina employing 15 -20 parts 11111111111666; part solidsin'said concentrated extract.

The process for producing mithramycin which comprisescultivatingStreptomyces argillaceils having the same identifying characteristics asATCC 12956, in

an aqueous nutrient, medium under agitated submerged, aerobic conditions,at a. temperature from about 24C. to 30C. fora period of from about oneday to about four days, until substantial antimicrobial activity' isimparted to such medium. i

10. The product of the process of claim 9. 11. A basic salt of thesubstance defined in claim 10.

l2 Th e processcla'imed in claim '9 wherein a substance selected fromthe group consisting of mithramycin A, mithramycin B and mithramycin Cis recovered from the fermentation broth by filtering the broth,adjusting the filtrate to a pH of about 2, extracting the filtrate withmethyl isobutyl ketone. concentrating the extract, absorbing theconcentrate on acid-washed alumina and cluting the mithramycin therefromwith 95 percent chloroformmethanol.

13. The process claimed in claim 9 wherein a substance selected from thegroup consisting of mithramycin A, mithramycin B and mithramycin C isrecovered from the fermentation broth by filtering the broth, adjustingthe filtrate to a pH of about 6. extracting the filtrate with n-butanol,concentrating the extract. allowing the concentrated extract to stand toeffect crystallization of a portion of said substance. removing thecrystals by filtration. concentrating and acidifying the filtrate,extracting the filtrate with ethyl acetate, concentrating the extract.absorbing said substance from the extract on acid-washed aluminaemploying l5-20 parts alumina per part solids in said concentratedextract and eluting the mithramycin therefrom with ethyl acetatefollowed by l percent ethanol in ethyl acetate.

14. A process for separating mithramycin A as defined in claim I andmithramycin B as defined in claim 3 from the fermentation brothcontaining same, which comprises contacting said broth with ethylacetate a solid mixture of two parts by weight silicic acid and one partby weight cellulose powder having the mixture of mithramycin A andmithramycin B absorbed thereon, whereby mithramycin A is selectivelyeluted therefrom, and thereafter contacting said solid mixture withethyl acetate containing 5 percent by volume of methanol. wherebymithramycin B is eluted therefrom.

15. A process for separating mithramycin A as defined in claim 1 andmithramycin C as defined in claim 16 from the fermentation brothcontaining same, which comprises adsorbing the mixture of mithramycin Aand mithramycin C on an adsorbent selected from the group consisting ofacid washed alumina and activated magnesium silicate. the amount of acidwashed alumina being 20-30 parts per part solids in said mixture, thentherefrom.

1. MITHRAMYCIN A, A ACIDIC SUBSTANCE WHICH RELATIVELY SOLUBLE IN WATER,LOWER ALCHOLS, ACETONE, ETHYL ACETANE AND METHYL ISOBUTYL DETONE,SLIGHTLY SOLUBLE IN DIETHYL ETHER AND BENZENE, AND SUBSTANTIALLYINSOLUBLE IN PETROLEUM ETHER, CARBON TETRACHLORIDE AND CYCLOHEXANE,WHICH IS CAPABLE OF FORMING SALTS WITH BASES, THE PURE CRYSTALLINEDIHYDRATE OF WHICH IS YELLOW AND CONTAINS THE ELEMENTS CARBON, HYDROGEN,AND OXYGEN IN SUBSTANTIALLY THE FOLLOWING PERCENTAGES BY WEIGHT: CARBON55.5%, HYDROGEN 7.2%, OXYGEN 37.3% (BY DIFFERENCE) AND HAS THE MOLECULRFORMULA C52H76O24.2H2O, WHICH DISPLAYS ULTRAVIOLET ABSORPTION MAXIMA INMETHANOL SOLUTION AT 230 AND 278 MP, WHICH HAS AN EQUIVALENT WEIGHT OFSUBSTANTIALLY 1115+$15 AND A PKA VALUE OF 7.6$0.2, WHICH EXHIBITS A DULLYELLOW FLOURESCENCE WHEN IRRADIATED WITH ULTRAVIOLET LIGHT, AND WHICH ASA 0.03 PERCENT DISPERSION IN POTASSIUM BROMIDE EXHIBITS ABSORPTIONMAXIMA IN THE INFRARED REGION OF THE SPECTRUM AT 736, 763, 803, 847,901, 950, 978, 1000, 1064, 1118, 1160, 1228, 1259, 1293, 1320, 1370,1370, 1396, 1440, 1500, 1580, 2626, 1720, 2850, 2910, 2950, 3390, CM-1,AND WHICH WHEN DISSOLVED IN METHANOL AT A CONCENTRATION OF 1 PERCENT HASTHE SPECIFIC ROTATION -58*, THE CRYSTALLINE ANHYDROUS FROM OF WHICH ISYELLOW, MELTS AT 184*-187*C. AND CONTAINS THE ELEMENTS CARBON, HYDROGENAND OXYGEN IN SUBSTANTIALLY THE FOLLOWING PERCENTAGES BY WEIGHT CARBON57.70% HYDROGEN 7.2% OXYGEN 35.1%, (BY DIFFERENCE) AND HAS THE MOLECULARFORMULA C52H76O24, AND WHICH DISPLAYS ULTRAVIOLET ABSORPTION MAXIMA INMETHANOL SOLUTION AT 230 AND 278MP AND WHICH AS AN 0.03 PERCENTDISPERSION IN POTASSIUM BROMIDE EXHIBITS ABSORPTION MAXIMA IN THEINFRARED REGION OF THE SPECTRUM AT 806, 852, 908, 952, 983, 1004, 1071,1126, 1170, 1236, 1266, 1300, 1330, 1374, 1515, 1631, 1724, 2890, AND3413 CM-1, AND WHICH ON THIN LAYER CHROMATOGRAPHY IN THE SYSTEMMETHANOL: ISOPROPANOL:METHYL ETHYL KETHONE (1:1:8) HAS AN RF VALUE OF0.6-0.7, THE CRYSTALLINE MAGNESIUM SALT OF WHICH IS BRIGHT YELLOW,DECOMPOSES AT 210*-215*C, HAS A PKA VALUE OF 4.5-4.7 AND AN EQUIVALENTWEIGHT OF 1110, AND CONTAINS THE ELEMENTS CARBON, HYDROGEN AND MAGNESIUMIS SUBSTANTIALLY THE FOLLOWING PERCENTAGES BY WEIGHT: CARBON 55.64%,HYDROGEN 7.19%, MAGNESIUM 1.15% AND HAS THE MOLECULAR FORMULA(C52H75O24)2MG, THE PURE CRYSTALLINE DIHYDRATED SODIUM SALT OF WHICH ISBRIGHT YELLOW AND CONTAINS THE ELEMENTS CARBON AND HYDROGEN INSUBSTANTIALLY THE FOLLOWING PERCENTAGES BY WEIGHT: CARBON 54.65%HYDROGEN 6.95% AND HAS THE MOLECULR FORMULA C22H75O24NA.2H2O, WHICHDESPLYS ULTRAVIOLET ABSORPTION MAXIMA IN METHANOL SOLUTION AT 240 285,315, AND 4525, MP AND WHEN DISSOLVED IN METHANOL AT A CONCENTRATION OF 1PERCENT HAS THE SPECIFIC ROTATION +38.8*.
 2. A basic salt of thesubstance defined in claim
 1. 3. Mithramycin B, an acidic substancewhich is highly soluble in water, lower alcohols, acetone, ethyl acetateand methyl isobutyl ketone, slightly soluble in diethyl ether andbenzene, and substantially insoluble in petroleum ether, carbontetrachloride, and cyclohexane, which is capable of forming salts withbases, the pure crystalline form of which is orange-yellow and containsthe elements carbon, hydrogen, and oxygen in substantially the followingpercentages by weight: CarbOn 55.22%, Hydrogen 7.13%, Oxygen 37.65%, (bydifference) which displays ultra-violet absorption maxima in methanolsolution at 230, 290, 320 and 410 m Mu , which exhibits a bright yellowfluorescence when irradiated with ultra-vioilet light, and which as a0.03 percent dispersion in potassium bromide exhibits absorption maximain the infrared region of the spectrum at 722, 743, 808,847, 903, 948,978, 1000, 1063, 1116, 1160, 1228, 1260, 1322, 1347, 1368, 1430, 1440,1500, 1585, 1630, 1720, 2850, 2900, 2940, 3380 cm
 1. 4. A basic salt ofthe substance defined in claim
 3. 5. Mithramycin C, an acidic substancesoluble in water, lower alcohols, ethyl acetate, acetone and methylisobutyl ketone, slightly soluble in diethyl ether and benzene, andsubstantially insoluble in petroleum ether, carbon tetrachloride andcyclohexane, which is capable of forming salts with bases, the purecrystalline form of which is bright yellow, melts at 182*-184* C. andcontains the elements carbon, hydrogen and oxygen in substantially thefollowing percentages by weight: Carbon 55.25%, Hydrogen 6.99%, Oxygen37.76%, (by difference) which displays ultra-violet absorption maxima inmethanol solution at 230 and 280 m Mu , and which as a 0.03 percentdispersion in potassium bromide exhibits absorption maxima in theinfrared region of the spectrum at 848, 905, 980, 1020, 1070, 1166,1233, 1266, 1299, 1408, 1515, 1634, 1706 and 3436 cm. 1, and which onthin layer chromatography in the system methanol:isopropanol:methylethyl ketone (1:1:8) has an Rf value of 0.15-0.25.
 6. A basic salt ofthe substance defined in claim
 5. 7. The process for producingmithramycin which comprises cultivating Streptomyces plicatus ATCC 12957in an aqueous nutrient medium under agitated submerged, aerobicconditions at a temperature of from about 24* to 30*C. for a period offrom about one day to about four days until substantial antimicrobialactivity is imparted to such medium.
 8. The process claimed in claim 7wherein a substance selected from the group consisting of mithramycin A,mithramycin B and mithramycin C is recovered from the fermentation brothby filtering the broth, adjusting the filtrate to a pH of about 6,extracting the filtrate with n-butanol, concentrating the extract,allowing the concentrated extract to stand to effect crystallization ofa portion of said substance, removing the crystals by filtration,concentrating and acidifying the filtrate, extracting the filtrate withethyl acetate, concentrating the extract, absorbing said substance fromthe extract on acid-washed alumina employing 15-20 parts alumina perpart solids in said concentrated extract.
 9. The process for producingmithramycin which comprises cultivating Streptomyces argillaceus havingthe same identifying characteristics as ATCC 12956, in an aqueousnutrient medium under agitated submerged, aerobic conditions, at atemperature from about 24*C. to 30*C. for a period of from about one dayto about four days, until substantial antimicrobial activity is impartedto such medium.
 10. The product of the process of claim
 9. 11. A basicsalt of the substance defined in claim
 10. 12. The process claimed inclaim 9 wherein a substance selected from the group consisting ofmithramycin A, mithramycin B and mithramycin C is recovered from thefermentation broth by filtering the broth, adjusting the filtrate to apH of about 2, extracting the filtrate with methyl isobutyl ketone,concentrating the extract, absorbing the concentrate on acid-washedalumina and eluting the mithramycin therefrom with 95 percentchloroformmethanol.
 13. The process claimed in claim 9 wherein asubstance selected from the group consisting of mithramycin A,mithramycin B and mithramycin C is recovered from the fermentation brothby filtering the broth, adjusting the filtrate to a pH of about 6,extracting the filtrate with n-butanol, concentrating the extract,allowing the concentrated extract to stand to effect crystallization ofa portion of said substance, removing the crystals by filtration,concentrating and acidifying the filtrate, extracting the filtrate withethyl acetate, concentrating the extract, absorbing said substance fromthe extract on acid-washed alumina employing 15-20 parts alumina perpart solids in said concentrated extract and eluting the mithramycintherefrom with ethyl acetate followed by 10 percent ethanol in ethylacetate.
 14. A process for separating mithramycin A as defined in claim1 and mithramycin B as defined in claim 3 from the fermentation brothcontaining same, which comprises contacting said broth with ethylacetate a solid mixture of two parts by weight silicic acid and one partby weight cellulose powder having the mixture of mithramycin A andmithramycin B absorbed thereon, whereby mithramycin A is selectivelyeluted therefrom, and thereafter contacting said solid mixture withethyl acetate containing 5 percent by volume of methanol, wherebymithramycin B is eluted therefrom.
 15. A process for separatingmithramycin A as defined in claim 1 and mithramycin C as defined inclaim 16 from the fermentation broth containing same, which comprisesadsorbing the mixture of mithramycin A and mithramycin C on an adsorbentselected from the group consisting of acid washed alumina and activatedmagnesium silicate, the amount of acid washed alumina being 20-30 partsper part solids in said mixture, then contacting said adsorbent withethyl acetate at a pH of about 2-3, whereby mithramycin A is selectivelyeluted therefrom, and thereafter contacting said adsorbent with ethylacetate containing up to 10-20 percent by volume of methanol, wherebymithramycin C is eluted therefrom.